A series of camptothecin open-ring lactone tripartate conjugates were synthesized, in which polyamine side chains with different architecture (ethane-1,2-diamine, spermidine, homospermidine, spermine, and 4,8,13,17-tetraza-icosane-1,20-diamine) are linked to the 21-carboxylic function through an amidic bond, while the 17-CH(2)OH is acetylated. The rationale for the synthesis of these compounds was to explore the influence of the polyamine architecture on the activity of these CPT conjugates into cells, since the positively charged ammonium cations would favor interaction through electrostatic binding to the negatively charged DNA backbone. Topoisomerase I-mediated DNA cleavage assay was used to investigate the ability of these compounds to stimulate the DNA damage. The cleavage pattern was found to be similar to that of SN38 for all the new CPTs. The CPT tripartates were tested for growth inhibition ability against the human non-small-cell lung cancer carcinoma NCI-H460 cell line. Although these compounds were less potent than topotecan, SN38, and CPT after 1 h of treatment, the antiproliferative effects greatly increased after 72 h of exposure. The growth inhibition potency during long-term exposure is correlated with the number of charges of the 21-amide substituent. Both cleavage assay and in vitro effects support the interpretation that the compounds may have inhibitory activity also in the open-ring form. The architecture of the polyamine moiety is important for antiproliferative activity, and a balance between the hydrophilic and lipophilic properties of the polyamine is critical for CPT potency.